Our specific objective with this fast-track STTR application is a diagnostic screen for the NIAID Biodefense Category B intestinal pathogens Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica in stool. These enteric protozoa share the characteristics of food and water-borne transmission, low infectious dose, and environmental stability. E. histolytica infections may be lethal, G. lamblia is most common, while Cryptosporidium spp. stands out as an important cause of chronic diarrhea without effective treatment in AIDS patients. The current lack of a low-cost screening fecal antigen test for enteric parasites compromises the healthcare of individuals presenting with diarrhea, as the only screening test available today is the stool O&P exam; a notoriously labor-intensive, non-specific and insensitive procedure. Our proposed diagnostic screen (the "Tri-Combo" ELISA) will provide cost-savings for clinical laboratories via the elimination from further testing specimens that are negative for all 3 parasites. The Tri-Combo ELISA is a conventional two-step ELISA format with HRP-conjugated detecting antibodies for colorimetric development. The unique multiplex nature of the test comes with its ability to simultaneously screen stool specimens for G. lamblia, Cryptosporidium spp. and E. histolytica using a single assay well. The Tri-Combo ELISA can be performed using visual interpretation of results for use in the field and in developing countries. At the same time, it is capable of conversion to automated platforms for use in reference laboratories and other large-scale surveillance scenarios; this will be useful with military and community water source outbreaks. Preliminary testing with the Tri-Combo ELISA indicates a >97% correlation to individual FDA-cleared ELISAs specific for G. lamblia, Cryptosporidium spp., and E. histolytica in stool with resolution by RT-PCR. Here, we propose optimization of the Tri-Combo ELISA in the R&D laboratory, followed by Good Manufacturing Practice (GMP) validation and FDA product clearance. Phase I will optimize the Tri-Combo ELISA, finalize GMP procedures, and prepare the clinical sites for evaluation. Phase II will complete the Tri-Combo ELISA manufacturing validation, establish performance characteristics at our consortium sites, and prepare FDA 510(k) pre-market clearance application documents. Innovation of the Tri-Combo ELISA lies with its ability to detect the three most common enteric protozoan parasites in the United States in a multiplexed format, and with its development process that allows three detection systems to be combined together in a single testing format. In the future, the process will be utilized to develop other multiplexed pathogen detection systems. [unreadable] [unreadable] A screening test is needed to detect the three most common intestinal protozoan parasites in the US (Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica). They are present in up to 10% of all stool samples submitted to reference laboratories, they are Category B Priority Biodefense Pathogens, and they share the characteristics of food and water-borne transmission, a low infectious dose, and environmental stability. Our objective is a diagnostic screen for the Giardia, Cryptosporidium, and E. histolytica in human stool specimens. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]